Project Summary/Abstract Long noncoding RNAs (lncRNAs) exhibit a great variety of functions ranging from gene expression regulation to mRNA processing and decay. Despite the critical roles lncRNAs play in numerous cellular pathways and their importance to cell viability, little is known about their functions at the molecular level. The human lncRNA, HOTAIR (2,148 nucleotides in length), has been heavily implicated in the development of cancer and is a predictor and stimulator of metastasis. HOTAIR has been suggested to act as a scaffold that recruits RNA-binding E3 ubiquitin ligase enzymes and their specific protein substrates, resulting in the ubiquitin-dependent degradation of these substrates. The molecular details on how HOTAIR biochemically stimulates the degradation of specific proteins are unknown. We hypothesize that HOTAIR recruits E3 ligase- substrate pairs to specific sites on the RNA and/or enzymatically stimulates ubiquitin ligase activity of the E3. Through a combination of in vitro biochemical assays and structure-function investigations using reconstituted protein-lncRNA complexes, this project aims to define the mechanistic contribution of HOTAIR in protein ubiquitination. For these studies, we have purified recombinant human E3 ubiquitin ligases and their respective substrates that were shown to interact with the HOTAIR RNA in vivo. In addition, we have successfully purified under native conditions, folded full-length HOTAIR and various domains of the RNA via in vitro transcription followed by gel filtration. Our preliminary studies show that full-length HOTAIR is able to stimulate autoubiquitination of the RNA-binding Dzip3 E3 ubiquitin ligase by an order of magnitude faster than in the absence of RNA. This suggests that HOTAIR might be involved in the regulation of E3 activity. Through the use of quantitative in vitro ubiquitination assays, we will define which structured RNA domains of HOTAIR are required for specific stimulation of Dzip3 autoubiquitination. In addition, we will reconstitute various Dzip3- HOTAIR complexes via chromatographic methods, and determine protein-RNA binding affinities using fluorescence polarization measurements. We will perform structure-function and mapping analyses to identify which protein and RNA domains lead to stable and active ribonucleoprotein complexes. In the long term, these complexes will also be investigated using structural approaches such as X-ray crystallography and cryo- electron microscopy. The overall goal of this study is to mechanistically determine how HOTAIR stimulates autoubiquitination activity of the Dzip3 E3 enzyme. In addition to the clear relevance of HOTAIR to human health, the results from these investigations will broaden our fundamental understanding of lncRNA biology and shed light on their molecular functions in enzymatic control.